The present invention provides plant growth-promoting activities of natural plant active constituent, calliterpenone and its derivatives belonging to the group phyllocladane diterpenoids. These phytochemicals and derivatives exhibit remarkable growth promoting activities on plant roots, shoots and promote seed germination both in monocotyledonous and dicotyledonous plants. These phytomolecules also possess antagonistic activity against allelochemicals of plant growth retardant nature and hence can be used to alleviate their detrimental effects.
Auxins and gibberellins are two main classes of natural compounds discovered simultaneously as plant growth stimulants. Besides these, cytokinins, abscisic acid, ethylene and many other compounds are also reported to possess plant growth regulatory properties. So far, more than 50 gibberellins have been discovered from natural sources. The growth stimulating activity has also been observed in several other types of natural plant constituents. Kaurenoic acid and steviol have been reported to be naturally occurring compounds having gibberellin like activity (Srivastava HS. 1999. Elements of Biochemistry. Rastogi Publications, Shivagi Road Merrut. Pp: 245-246). Helminthosporic acids isolated from Helminthosporium sativum and phaseolic acid isolated from the bean (Phaseolus species) seeds have also showed growth promoting activities. (Leopold A. C. and Kriedemann P. E. Plant Growth and Development Tata McGraw Hill Publishing Company Ltd., New Delhi. 137-140. 1981).
In recent years many papers and patents dealing with the plant growth promoting activities of natural and synthetic compounds have appeared. Important of them are salicilic acid (H S Gehlot et al. Advances in Plant Physiology Vol-I, 273-289, 1998), trioctanol(Ries et al, Science, 195 :1339, 1977) and other compounds reported from time to time. Goyal R. et al. reported three oxygenated alantolides from Inula racemosa (Phytochemistry 29(7) 2341-2343, 1990), three sesquiterpenoids, Saussureal, dehydrocostus lactone and costunolide isolated from Saussurea lappa have also shown growth promoting activities (I. P. Singh et al. Phytochemistry 31(7) 2529-2531, 1992 and K. K. Talwar et al. Phytochemistry 31 (1) 336-338 1992).
Among the synthetic derivatives, many compounds like triazoles, have been reported as a new group of promising plant growth promoters (R. P.Raghava et al. Advances in Plant Physiology, Vol-I 291-320, 1998). Various derivatives of organic acids reported to have plant growth promoting activity (e.g. West German Patent No. 1916054 discloses uses of xcex1-hydroxy and xcex1-ketoalkanoic acid having 7-10 carbon atoms and their derivatives; U.S. Pat. No. 3, 148,049 discloses growth promoting activity of certain halogenated keto acids under drought conditions). Mikami et al. (Agr Biol Chem 1970 3 4, 977-979) reported a number of hydroxy acids as plant growth regulators. Kamuro et al.(U.S. Pat. Nos. 6,093,683; 2000; 5,776,860; 1998; 4,846,877; 1989 and 6,271,176; 2001) described the derivatives of jasmonic acids as plant growth promoters. Danzig Morris J. et al. (U.S. Pat. No. 4,806,149; 1989) disclosed that monoxanthates and dixanthates of dipropylene glycol and triethylene glycol increase the growth and concentration of chlorophyll in plants.
Thus many active compounds have been reported so far to promote growth in plants. These compounds help in the increase in sizes of fruit, root, and shoot and in the total yield of the crop. However, it is difficult to obtain these compounds in great quantities from plants and cost to prepare them synthetically will be very high.
Callicarpa macrophylla Vahl. (Verbanaceae) is an erect shrub common to Indo-Gangetic plains and sub-Himalayan tracts from Kashmir to Assam ascending upto 2000 m. Leaves of plant are commonly used in rheumatism and aromatic root oil is given in stomach troubles. The plant has been investigated for its chemical constituents (Asolkar et al. Second supplement to Glossary of Indian Medicinal Plants with active principles, part 1. PID,CSIR, New Delhi, 155,1992).
The isolation of calliterpenone and its monoacetate from Callicarpa macrophylla was first reported by Chaterjee et al, Tetrahedron 28, 4319, 1972, and assigned the structure as 16xcex1, 17 dihydroxy-ent kauan-II-one and its 17-acetate. Based on chemical and spectroscopic evidences, these structures were however, revised as 16xcex1, 17 dihydroxy phyllocladan-3one and its 17-acetate on the basis further chemical xe2x80x98H NMR, NOE and LIS and X-ray crystallographic data studies by Ahmad and Zaman (Tetrahedron Lett. 2179,1973), Fujita et al (Phytochemistry 14 1975 2249) and Wong et al (Acta Crystallography Sec. C 47 1991 906). Later, this compound was also isolated from some other species of the genus Callicarpa.
During previous studies on Callicarpa macrophylla, natural compounds 16xcex1, 17 dihydroxy phyllocladan-3 one (calliterpenone), its 17-acetate (calliterpenone monoacetate) and two related minor constituents 16xcex1, 17-isopropylidino 3-oxo phyllocladane (isopropylideno calliterpenone) and 30xcex2, 16xcex1, 17. trihydroxy phyllocladane (trihydroxy calliterpenone) werwe isolated and diacetate derivative of calliterpenone also prepared (Singh et al. Phytochemistry 37, 1994,587 and Indian Jour. of Chem.33B, 1994 1205). The complete spectral assignment of all the 5 phllocladane diterpenoids have also been done (Agarwal et al. Ind. Jour. of Chem.35B. 1996. 803-805).
The basis of the present invention is the finding that these pyllocladane diterpenoids when used in particular concentrations behave as growth promoters. Some of these compounds on certain dilutions produce more growth enhancement than that produced by GA3 (Gibberellic acid) in identical conditions.
The invention also provides novel growth promoting activity of phyllocladane diterpenoids like calliterpenone and derivatives isolated from plants of Callicarpa macrophylla and their antagonizing effects against plant growth retardants.
Further the invention also provides the effective amount of phyllocladane diterpenoids selected from calliterpenone, calliterpenone mono-acetate, calliterpenone di-acetate, isopropylidino calliterpenone and tryhydroxy calliterpenone wherein 0.001 to 0.1 xcexcM (w/v) concentrations of the said compounds can be used as sprays or in the media in tissue cultures or soaking of seeds, roots, shoots etc in said solutions.
The said solutions are prepared by dissolving required amount in minimum amount of water/organic solvents like ethyl alcohol, methanol, propenol followed by gentle warming and then the desired concentrations are achieved by adding suitable amount of distilled water.
The present invention describes the growth promoting activities of phyllocladane diterpenoids isolated from the leaves of plant Callicarpa macrophylla that are not only cheap but show better activity at a particular dilution than the well known growth promoting hormone Gibberellic Acid (GA3). In our experiment calliterpenone, its monoacetate and certain other derivatives naturally isolated from the plant Callicarpa macrophylla Vahl. (Verbanaceae) have shown promising growth promoting activities.
Calliterpenone(16xcex1, 17 dihydroxy phyllocladan-3 one),a phyllocladane diterpenoid, is a stereoisomer of abbeokutone (16,17 dihydroxy kauran-3-one). Abbeokutone has kauranoid skeleton while calliterpenone has phyllocladane skeleton. The antagonistic activity of these phytoderivatives against negative allelochemicals makes their utility much higher as plant growth supporting chemicals.
As noted above the activity of phyllocladane diterpenoids used in the practice of this invention was observed when these compounds were tested for their effects on seed germination, root and shoot growth of certain plant species and their antagonistic effect on plant growth retardants by the method described by Bagchi G. D. et al Phytochemistry, 45(6)1131-1133, 1997.
Since the assay involves growing the plants/seedlings in aqueous solution, it demonstrates the usefulness of the process in promoting the growth of plants in hydroponics culture.
Like wise the process of this invention is also useful when plants are propagated by tissue culture or the solutions are sprayed on the plants. Further, these compounds may be utilized to antagonize the allelochemical effects produced by the preceding crops.
Phyllocladane diterpenoids of present invention are represented by general formula
The phyllocladane diterpenoids used in the process of present invention are seen to produce more than one type of growth regulatory effects on the plants. This effect on a plant depends on a number of variables including type of phyllocladane diterpenoid compounds used, their concentration, type of the plant species and the effective amount of the compound needed to obtain the desired response. The most suitable concentration for a particular application is readily determined by screening test as given in examples. Most, preferred phyllocladane diterpenoid are calliterpenone and calliterpenone mono-acetate and preferred concentration is 0.01 xcexcM-0.00 xcexcM.
The activity of phyllocladane diterpenoid used in practice of this invention was discovered when they were tested in two monocotyledonous and two dicotyledonous plants. Using general testing procedure as described earlier (Bagchi G. D. et al Phytochemistry, 45(6): 1131-1133, 1997). The test showed that different phyllocladane diterpenoids have growth promoting activities when used in concentrations between 0.1 xcexcM and 0.001 xcexcM (w/v basis), while on increasing the concentration, growth inhibitory activity was observed in certain cases.
For phyllocladane diterpenoid the optimum growth promoting concentration was 0.00 xcexcM. The phyllocladane diterpenoids are generally useful in the practice of this invention are calliterpenone, calliterpenone mono-acetate, calliterpenone di-acetate, isopropylideno calliterpenone and trihydroxy calliterpenone. Solutions of the phyllocladane diterpenoids are conveniently applied to the plant in water added to the plant which may be prepared by dissolving the required amount in minimum amount of water/organic solvents like acetone or ethyl alcohol, methanol, propenol and followed by gentle warming and then the desired concentrations may be achieved by adding suitable amount of distilled water. Nutrients required by the plants may be added in the water. The solution of phyllocladane diterpenoid may be sprayed or roots, stems and seeds etc. may be soaked in it.
Phyllocladane diterpenoids, calliterpenone and calliterpenone monoacetate were obtained from the plant Callicarpa macrophylla, as described earlier (Singh and Agarwal. Phytochemistry 32(2) 587-588, 1994). other phyllocladane diterpenoids may be isolated from the plants or may be transformed chemically or microbially from naturally isolated calliterpenone (A. K.Singh and P. K.Agarwal. Indian Journal of Chemistry 33(B) 1205-1205,1994).
The invention is illustrated by following examples, which should not be constructed to limit the scope of present invention. The concentrations are given in micromolar solution, weight/volume basis.
Plant growth promoting activity of calliterpenone, calliterpenone monoacetate, and calliterpenone di-acetate was examined at 0.1, 0.01 and 0.001 xcexcM concentrations. The tests were conducted against seeds of two monocotyledonous (Hordeum vulgare and Triticum aestivum) and two dicotyledonous plants (Vigna radiata and Trigonella foenum-graecum). Control seeds were germinated in distilled water and all the observations were compared with GA3 grown seedlings. The root and shoot growth of all the plant species were affected by the phyllocladane diterpenoids, although the extent of effect varied considerably in the plant species with the chemicals and their concentrations. In general, the growth promoting activities were more pronounced with dilute solutions, while the higher concentrations proved toxic to the species tested. Details of the procedure adopted and results obtained are discussed in the various examples.